Human Collagen Prolyl 4-Hydroxylase Is Activated by Ligands for Its Iron Center.

Department of Biochemistry and ‡Department of Chemistry, University of Wisconsin-Madison , Madison, Wisconsin 53706, United States.

Abstract

Collagen is the most abundant protein in animals. The posttranslational hydroxylation of proline residues in collagen contributes greatly to its conformational stability. Deficient hydroxylation is associated with a variety of disease states, including scurvy. The hydroxylation of proline residues in collagen is catalyzed by an Fe(II)- and α-ketoglutarate-dependent dioxygenase, collagen prolyl 4-hydroxylase (CP4H). CP4H has long been known to suffer oxidative inactivation during catalysis, and the cofactor ascorbate (vitamin C) is required to reactivate the enzyme by reducing its iron center from Fe(III) to Fe(II). Herein, we report on the discovery of the first synthetic activators of CP4H. Specifically, we find that 2,2'-bipyridine-4-carboxylate and 2,2'-bipyridine-5-carboxylate serve as ligands for the iron center in human CP4H that enhance the rate of ascorbate-dependent reactivation. This new mode of CP4H activation is available to other biheteroaryl compounds but does not necessarily extend to other prolyl 4-hydroxylases. As collagen is weakened in many indications, analogous activators of CP4H could have therapeutic benefits.

Activation of CP4H1 by bipy4C and bipy5C is dose-dependent and requires the Vitamin C cofactor. (A) Dose–response curves for activation (and inhibition) of CP4H1 by bipymonoCs. CP4H assays were performed in the presence and absence of activator (0.3–3000 μM) and ascorbate (2 mM), and in the presence of a saturating concentration of α-ketoglutarate (1 mM). Relative activity values are the mean (±SE) of three independent experiments) and represent the ratio of enzymatic activity in the presence and absence of activator. (B) Dependence of the activation observed by bipy4C and bipy5C on ascorbate concentration. CP4H assays were performed in the presence and absence of activator (100 μM) and ascorbate (20 μM–2 mM), and in the presence of 1 mM α-ketoglutarate. Relative activity values are the mean (±SE) of three independent experiments) and represent the ratio of CP4H activity observed in the presence and absence of activator.

Putative mechanism of catalysis by CP4H. O2 and the peptide substrate bind to a CP4H•Fe(II)•α-ketoglutarate complex in an ordered fashion. Oxidative decarboxylation of α-ketoglutarate leads to the formation of a highly reactive Fe(IV)=O species (ferryl ion) that hydroxylates the peptide substrate via a radical rebound process.- For prolonged activity, CP4H requires VitCred during an “uncoupled reaction” in which CP4H decarboxylates α-ketoglutarate in the absence of the peptide substrate. The ferryl ion decays to an inactive Fe(III) state, and VitCred reduces the iron center back to the requisite Fe(II) state., We propose that bipy4C and bipy5C activate CP4H1 during this uncoupled reaction by serving as a ligand that enhances the rate of the VitCred-dependent reduction of the iron center.

Iron-binding properties of bipy4C and bipy5C. (A) Absorption spectra of activators (300 μM) in the presence and absence of Fe(II)SO4 (100 μM). Complexes of bipy4C and bipy5C with Fe(II) showed local absorption maxima (λmax) of 533 nm and 544 nm, respectively. (B) Titrations of Fe(II) with bipy4C and bipy5C. Values of Fe20-EC50 were (33 ± 0.8) μM and (43 ± 2) μM, respectively. (C) Job’s plots for the complexes of Fe(II) with bipy4C and bipy5C. Values of χ at the point intersection were 0.75 ± 0.02 and 0.76 ± 0.06, respectively, consistent with the formation of Fe(ligand)3 complexes.

Structure–activity relationships of bipymonoCs and related chemical analogues for the activation of human CP4H1. (A) Chemical structures and abbreviations. (B) Screen (10 μM) of the compounds depicted in panel A against human CP4H1. CP4H1 assays were performed in the presence of 100 μM α-ketoglutarate. Relative activity values are the mean (±SE) of three independent experiments and represent the ratio of CP4H1 activity observed in the presence and absence of the activator or chemical analogue.

Preincubation of bipy4C with iron abolishes activation of CP4H1. CP4H1 assays were performed in the presence of 100 μM α-ketoglutarate and 10 μM bipy4C that was either added last (“Standard Assay”) or preincubated with Fe(II)SO4 prior to addition to the assay. Relative activity values are the mean (±SE) of three independent experiments and represent the ratio of CP4H activity observed in the presence and absence of activator.

Activation of CP4H1 by bipymonoCs is competitive with succinate. CP4H1 assays were performed in the presence of 100 μM activator and 1 mM α-ketoglutarate. Relative activity values are the mean (±SE) of three independent experiments and represent the ratio of CP4H activity observed in the presence and absence of the activator.

Inhibition or activation of CP4H1 by biheteroarylcarboxylates and related analogues is dependent upon α-ketoglutarate and succinate. CP4H1 assays were performed in the presence of 2 mM ascorbate. Relative activity values are the mean (±SE) of three independent experiments and represent the ratio of CP4H activity observed in the presence and absence of a modulator. (A) Effect of added succinate and α-ketoglutarate on CP4H1 activity. (B) Effect of bipy on CP4H1 activity. (C) Effect of 25PDC on CP4H1 activity and its dependence upon α-ketoglutarate and succinate. (D, E, F) Effect of bipy55′DC (D), bipy45′DC (E), and pythiDC (F) on CP4H1 activity and their dependence upon α-ketoglutarate and succinate.

Activation of prolyl 4-hydroxylases by bipymonoCs. Human CP4H1, human PHD2, and C. reinhardtii P4H-1 (CrP4H-1) were assayed in the presence or absence of bipy4C (A) or bipy5C (B) at 10 μM. Relative activity values are the mean (±SE) of three independent experiments and represent the ratio of CP4H activity observed in the presence and absence of the activator in question.